This invention relates to the detection of patients at risk for developing integrin antagonist/agonist mediated disease states. This invention relates to assays useful for the detection in a patient bodily fluid sample of drug-dependent antibodies which bind to integrins, or intergrin-associated proteins or complexes thereof in the presence of an integrin antagonist/agonist. This invention also relates to assays useful for the detection in a patient bodily fluid sample of drug-dependent antibodies (DDABs) that bind to integrins, including the platelet glycoprotein IIb/IIIa (GPIIb/IIIa), in the presence of a integrin agonist and/or antagonist. This invention also relates to procedures for identifying integrin antagonists/agonists that are less prone to elicit integrin antagonist/agonist mediated disease states.
This invention also relates to procedures which increase the recovery of integrin-directed antibodies in body fluids, resulting in an increased sensitivity and specificity of DDAB detection assays. This invention also relates to procedures for treating blood samples, which dissociate antibodies to GPIIb/IIIa from the platelet surface, thereby increasing the recovery from the platelet supernatant. This invention also relates to the use of different GPIIb/IIIa preparations to identify patients at risk for early-onset thrombocytopenia upon treatment with GPIIb/IIIa antagonist/agonists, thereby increasing the specificity of antibody detection.
This invention also relates to the use of DDABs as a positive control and calibration standard for DDAB assays.
Such methods, procedures and assays are useful for identifying patients who may be at risk to develop disease states mediated by treatment with integrin antagonists/agonists.
Thromboembolic diseases, including stable and unstable angina pectoris, myocardial infarction, stroke and lung embolism, are the major cause of disability and mortality in most developed countries. Recently, therapeutic strategies aimed at interfering with the binding of ligands to the GPIIb/IIIa integrin have been explored to treat these patient groups. Platelet GPIIb/IIIa is the main platelet receptor for fibrinogen and other adhesive glycoproteins, including fibronectin, vitronectin and von Willebrand factor. Interference of ligand binding with this receptor has been proven beneficial in animal models of thromboembolic disease (Coller, B. S. GPIIb/IIIa Antagonists: Pathophysiologic and Therapeutic Insights From Studies of C7E3 FAB. Thromb. Haemost. 78: 1, 730-735, 1997), and in limited studies involving human subjects (White, H. D. Unmet Therapeutic Needs in the Management of Acute Ixchemia. Am. J. Cardiol. 80: 4A, 2B-10B, 1997; Tcheng, J. E. Glycoprotein IIb/IIIa Receptor Inhibitors: Putting EPIC, IMPACT II, RESTORE, and EPILOG Trials Into Perspective. Am. J. Cardiol. 78: 3A, 35-40, 1996).
A number of cell surface receptor proteins, referred to as integrins or adhesion protein receptors, have been identified which bind to extracellular matrix ligands or other cell adhesion protein ligands thereby mediating cell-cell and cell-matrix adhesion processes. The integrins are encoded by genes belonging to a gene superfamily and are typically composed of heterodimeric transmembrane proteins containing xcex1- and xcex2-subunits. Integrin subfamilies contain a common xcex2-subunit combined with different xcex1-subunits to form adhesion protein receptors with different specificities. In addition to GPIIb/IIIa, a number of other integrin cell surface receptors have been identified. For example, members of the xcex21 subfamily, xcex14xcex21 and xcex15xcex21, have been implicated in various inflammatory processes, including rheumatoid arthritis, allergy, asthma and autoimmune disorders.
The integrin GPIIb/IIIa, also referred to as the platelet fibrinogen receptor, is the membrane protein mediating platelet aggregation. GPIIb/IIIa in activated platelets is known to bind four soluble RGD containing adhesive proteins, namely fibrinogen, von Willebrand factor, fibronectin, and vitronectin. The term xe2x80x9cIRGDxe2x80x9d refers to the amino acid sequence Arg-Gly-Asp. The binding of fibrinogen and von Willebrand factor to GPIIb/IIIa causes platelets to aggregate. The binding of fibrinogen is mediated in part by the RGD recognition sequence which is common to the adhesive proteins that bind GPIIb/IIIa. RGD-peptidomimetic GPIIb/IIIa antagonist compounds are known to block fibrinogen binding and prevent platelet aggregation and the formation of platelet thrombi. GPIIb/IIIa antagonists represent an important new approach for anti-platelet therapy for the treatment of thromboembolic disorders.
Approximately 1% of individuals receiving certain GPIIb/IIIa antagonists develop life-threatening thrombocytopenia. The principal cause of these thrombocytopenias is thought to be immune mediated, due to the presence of drug-dependent anti-platelet antibodies (Berkowitz, S. D., Harrington, R. A., Rund, M. M., and Tcheng, J. E. Acute Profound Thrombocytopenia After C7E3 FAB (abciximab) Therapy. Circulation 95:809-813, 1997). However, such drug-dependent anti-platelet antibodies have not been found in all patients undergoing GPIIb/IIIa inhibitor treatment, leading to speculation that there may be other causes for GPIIb/IIIa-inhibitor-dependent thrombocytopenia.
The general phenomenon of drug-dependent thrombocytopenia/thromboembolic complications is well known. Clinically important examples are heparin-induced thrombocytopenia (HIT) (Amiral, J., Bridley, F., Wolf, M., et al., Antibodies to macromolecular platelet factor IV-heparin complexes in heparin-induced thrombocytopenia: A study of 44 cases. Thromb. Haemost. 1995, 73:21-28; Ansell, J., Deykin, D., Heparin-induced thrombocytopenia and recurrent thromboembolism. Am. J. Hematol. 1980, 8:325-332), and heparin-induced thrombotic thrombocytopenia (HITT), though many other drugs have been implicated (Kelton, J. G., Sheridan, D. P., Santos, A. V., et al. Heparin-induced thrombocytopenia: Laboratory studies. Blood, 1988, 72:925-930; Chong, B., Berndt, M. Heparin induced thrombocytopenia. Blut 1989, 58:53-57; Curtis, B. R., McFarland, J. G., Wu, G-G., Visentin, G. P., and Aster, R. H., Antibodies in sulfonamide-induced immune thrombocytopenia recognize calcium-dependent epitopes on the glycoprotein IIb/IIIa complex. Blood, 1994 84:176-183). HIT and HITT are thought to be of immune origin involving binding to the platelet of drug-dependent anti-platelet antibodies induced by the formation of heparin/platelet Factor IV/antibody complexes (Karpatikin, S., Drug-induced thrombocytopenia. 1971, Amer. J. Medical Sciences, 262:68-78). Platelet clearance is thought to be mediated by the reticuloendothelial system (RES). In some cases such drug/antibody complexes are reported to activate platelets, leading directly to platelet secretion and aggregation (Amiral, J., wolf, M., Fisher, A. M., Boyer-Neumann, C., Vissac, A. M., and Meyer, D. Pathogenicity of IgA and/or IgM antibodies to heparin-platelet Factor IV complexes in patients with heparin-induced thrombocytopenia. British J. of Haem. 1996, 92:954-959).
Cases of thrombocytopenia of unknown origin are referred to as idiopathic thrombocytopenic purpura (ITP). In most patients this disorder is thought to be caused by autoantibodies against platelet membrane glycoproteins (Gonzalez-Conejero, R., Rivera, J., Rosillo, M. C., Lozano, M. L., and Garcia, V. V., Comparative study of three methods to detect free plasma antiplatelet antibodies. Acta Haematol., 96:135-139, 1996; Stockelber, D., Hou, M., Jacobson, S., Kutti, J., Wadenvik, H., Detection of platelet antibodies in chronic idiopathic thrombocytopenic purpura (ITP). A comparative study using flow cytometry, a whole platelet ELISA, and an antigen capture ELISA. Eur. J. Haematol., 56:72-77, 1996) and possibly glycolipids (Arnout, J. The pathogensis of the antiphospholipid syndrome: A hypothesis based on parallelisms with heparin-induced thrombocytopenia. Thrombosis and Haemostasis, 75:536-541, 1996; Cuadrado, M. J., Mujic, F., Munoz, E., Khamashta, M. A., Hughes, G. R. V., Thrombocytopenia in the antiphospholipid syndrome. Annals of the Rheumatic Diseases, 56:194-196, 1997), with removal of IgG-sensitized platelets by the RES.
GPIIb/IIIa antagonist-dependent drug-dependent antibodies (DDABs) are defined here as antibodies that (a) bind to platelets in the presence of a GPIIb/IIIa antagonist but do not bind to platelets in the absence of a GPIIb/IIIa antagonist, or (b) which bind to platelets in the absence of a GPIIb/IIIa antagonist, but whose binding or ability to induce platelet activation is potentiated by GPIIb/IIIa antagonists.
GPIIb/IIIa DDABs may bind, for example, to stable neoepitopes in GPIIb/IIIa and/or GPIIb/IIIa-associated proteins or complexes, which are mediated or induced by the binding of the GPIIb/IIIa antagonist to GPIIb/IIIa. The DDABs may also bind to unstable neoepitopes requiring the constant presence of both GPIIb/IIIa and/or GPIIb/IIIa-associated proteins or complexes, and the antagonist, or to structural entities consisting of GPIIb/IIIa and/or GPIIb/IIIa-associated proteins or complexes, and the antagonist/agonist itself.
The complications associated with the use of GPIIb/IIIa antagonist/agonists may severely limit their use, and integrin antagonist/agonists in general, because patients may develop a thrombocytopenic episode mediated by DDABs and/or other drug-dependent mechanisms.
It follows from the foregoing considerations that a sensitive and specific assay that can detect such GPIIb/IIIa directed DDABs may be beneficial in identifying patients with DDABs which are present prior to treatment with the GPIIb/IIIa antagonist, and/or antibodies which develop and increase in titer following administration of the GPIIb/IIIa antagonist. Patients with pre-existing or developing DDAB titer may have a greater risk of undergoing thrombocytopenic episodes following administration of the GPIIb/IIIa antagonist. Patients which are determined to have pre-existing DDABs may either be excluded from therapy with GPIIb/IIIa antagonists, or may be treated with a compound which is less prone to potentiate the binding of DDABs. Alternatively, if a DDAB titer should develop, the therapy can be stopped prior to the onset of a clinically significant thrombocytopenic episode. Patients with pre-existing DDABs may be at risk of developing a thrombocytopenic episode upon treatment with GPIIb/IIIa antagonist.
Low titers of pre-existing DDABs may be present in a relatively large percentage of the general population. It follows that procedures aimed at identifying patients in the DDAB-positive population that are at increased risk for thrombocytopenia/thromboembolic complications will facilitate the exclusion of this xe2x80x9chigh riskxe2x80x9d population from therapy with a specific GPIIb/IIIa antagonist, treatment with chemically distinct GPIIb/IIIa antagonists, or identify patients in need of extensive monitoring during treatment. The use of specific conformers of GPIIb/IIIa (for example, RGD retained and non-retained) for the identification of patients with a high propensity to develop early-onset thrombocytopenia/thromboembolic complications has not been taught in the art.
In patients with developing or increasing DDAB titer, the identification of such an increase at the earliest time point is necessary to terminate therapy with a specific GPIIb/IIIa antagonist prior to the development of a clinically significant thrombocytopenic episode. A number of procedures aimed at recovering platelet associated antibodies are known in the art. They require isolation of platelets from whole blood and treatment with low or high pH, or protein denaturants. These procedures can only be performed in specialized laboratories on freshly prepared biological specimens. In addition, false-negative results are to be expected due to inherent instabilities of specific antibodies, excluding a reliable functional analysis of the resulting platelet eluate. Ethylenediaminetetraacetic acid (EDTA) treatment of isolated platelets has been reported to dissociate the GPIIb/IIIa complex, and reduced binding of conformationally sensitive murine antibodies to GPIIb/IIIa has been observed. The use of EDTA treatment in whole blood using human autoantibodies to GPIIb/IIIa or DDABs directed to GPIIb/IIIa has not been reported. In addition, the combined treatment with thrombin receptor activating peptides or other platelet agonists and EDTA has not been taught in the art.
The utility of assays aimed at detecting DDABs can be increased if reliable DDAB standards are available. The standard should be reactive with the same secondary antibody detection system as the human DDAB and thus allow for a calibration of the experimental results. The method and composition of such a standard has not been taught in the art.
There remains the need for sensitive, specific and easy-to-use assays to be used in conjunction with integrin antagonist/agonist treatment, such assays being capable of detection of low levels of integrin antagonist/agonist DDABs which may be present in an individual prior to the administration of an integrin antagonist/agonist and/or for the detection of developing DDABs following treatment with the integrin antagonist/agonist. The present invention provides such assays for the detection of integrin antagonist/agonist DDABs. There is a continuing need to increase the sensitivity, specificity, and ease of use of methods to detect autoantibodies and DDABs to integrins. The present invention provides such procedures for the detection of integrin-directed antibodies.
Savoca et al., U.S. Pat. No. 4,717,654 describes a method for the detection of DDABs associated with thrombocytopenia. The disclosed method requires platelets and does not utilize purified GPIIb/IIIa. Aster et al., U.S. Pat. No. 5,585,243 describes an assay to determine which drug causes cytopenia when the patient receives multiple drugs.
The present invention differs in that it is more sensitive. The present invention is capable of detecting DDABs of far lower platelet binding than reported in the art. In addition, the present invention differs from the existing art in that it is a method for the specific detection of such DDABs which might be responsive to integrin antagonists/agonists in general, and to GPIIb/IIIa antagonists in particular.
This invention provides treatment methods and procedures to identify patients at risk for integrin antagonist/agonist mediated disease states. The present invention provides assays and methods useful for the detection, in a patient bodily fluid sample, of drug-dependent antibodies that bind to cells in the presence of an integrin antagonist/agonist. The present invention provides sensitive, specific and easy-to-use assays which may be used in conjunction with integrin antagonist/agonist treatment. These assays are capable of detection of low levels of integrin antagonist/agonist-dependent antibodies that bind to cells which may be present in an individual prior to the administration of an integrin antagonist/agonist, and/or for the detection of developing integrin-antagonist/agonist-dependent substances following treatment with the integrin antagonist/agonist.
An object of the present invention provides assays and methods for the detection in a patient bodily fluid sample of DDABs which bind to platelets in the presence of a GPIIb/IIIa antagonist. The present invention provides an enzyme-linked immunosorbent assay (ELISA) using purified immobilized GPIIb/IIIa and certain GPIIb/IIIa antagonists in the assay. The GPIIb/IIIa DDAB ELISA of the present invention detects pre-existing GPIIb/IIIa DDABs (for example, DDABs which are pre-existing in the patient prior to the patient being administered the GPIIb/IIIa antagonist). The GPIIb/IIIa DDAB ELISA of the present invention also detects GPIIb/IIIa DDABs for which an antibody titer develops following the GPIIb/IIIa antagonist being administered to the patient, such GPIIb/IIIa DDABs being potentiated by the presence of the GPIIb/IIIa antagonists. The present assays and methods may be used to identify individuals having GPIIb/IIIa antagonist-induced DDABs and may be used to exclude, terminate, and/or change therapeutic modalities with GPIIb/IIIa antagonists prior to the onset of thrombocytopenia/thromboembolic complications.
Another object of the invention provides procedures for the increased recovery of integrin directed antibodies from cell surfaces. As a result, the sensitivity and specificity of assays aimed at detection of these antibodies is increased. The procedure entails treatment of cells with strong chelating agents, including EDTA, for extended periods of time at different temperatures, resulting in the dissociation of the integrin-directed antibodies from the cell surface and their recovery in a biologically active form in the supernatant after separation of the cellular components from the fluid phase. Such treatment may be performed ex vivo in whole blood or fractions thereof, or on tissue samples. The resulting samples may then be analyzed by assays capable of detecting integrin-directed antibodies.
It has been found in the present invention that use of different GPIIb/IIIa antagonists in the GPIIb/IIIa DDAB ELISA detect different DDABs. Thus, different GPIIb/IIIa antagonists in the GPIIb/IIIa DDAB ELISA differ in their ability to induce the formation of epitopes which are recognized by DDABs in a patient. Thus the present assays may be employed to identify integrin antagonists/agonist which may be less likely to induce DDABs or induce epitopes which are recognized by pre-existing or developing DDABs.
This invention also relates to the use of different GPIIb/IIIa preparations to identify patients at risk for early-onset thrombocytopenia/thromboembolic complications upon treatment with GPIIb/IIIa antagonists, thereby increasing the specificity of antibody detection.
This invention also relates to the use of DDABs and humanized chimeric antibodies as a standard for DDAB assays.
The present assays may be used to identify patients at risk of developing GPIIb/IIIa antagonist-induced thrombocytopenia or thromboembolic complications and/or to identify patients who are not at risk of developing GPIIb/IIIa antagonist-induced thrombocytopenia or thromboembolic complications.